to be useful m conversron reactrons. The eluates are stored on Ice and, other
than radroactrve decay, are often stable for weeks.
The Cell-free Conversion Reaction
3.1 2.
The converston of 35S-PrP-sen to protemase K-resrstant forms can be
achieved by incubating 35S-PrP-sen with brain-derived PrP-res that has been
pretreated with 0-3M guanidme hydrochlorrde (GdnHCl) at 37В°C for 0.25-24 h
(Fig. 1) (52-54). The optrmal GdnHCl concentration varies slightly with the
PrP-res preparatron. For instance, the converting actrvtty of many preparations
of hamster PrP-res (263K strain) 1senhanced by a 3M GdnHCl pretreatment
(52), whereas the activity of some other preparations of 263K PrP-res have
been optima1 with pretreatments of 2-2SMGdnHCl. The reason for this varia-
tion in GdnHCl sensitivity is unclear but it may be influenced in part by the
purity of the preparation or repeated freeze-thaw cycles. A l-3 ug/pL suspen-
sion of PrP-res in the desired GdnHCl concentratron IS mixed with an equal
volume of 35S-PrP-seneluate prediluted to the same GdnHCl concentratron.
The mixture is then diluted further to 0.75M GdnHCl using TN supplemented
with cetylpyridinium chloride (CPC) to give a final concentration of 1.5 mM
CPC and incubated at 37В°C for 216 h.
The PK-resistance of the 35S-PrPis then tested after a further dilution of the
GdnHCl to 0.075 - 0.4M by digesting with 20-500 ug/mL of PK for 1 h at
37OC (54). The PK IS inhibited using Pefabloc SC (Boehringer Mannheim) as
recommended by the manufacturer. Twenty mrcrograms of a carrier protein
(thyroglobulin) is added, and the proteins are precipitated with methanol. The
294 Caughey et al.
resulting pellet IS boiled m SDS-PAGE sample buffer and electrophoresed.
Radiolabeled PrP-res was visualized by fluorography wtth Entenslfy (DuPont,
Wilmington, DE) or Phosphorlmager analysis (Molecular Dynamics, Sunny-
vale, CA)
In summary, a typical reaction would be*
I 2 pL of 1 mg/mL hamster PrP-res m 3MGdnHCl
2 2 pL hamster 35S-PrP-sen with 30,000 cpm/pL m 3M GdnHCl
3 12pLTN+CPC
4 MIX these constituents, somcate bnefly, and Incubate for 1 d at 37В°C
5 Dilute with 64 pL TN and then add 4 pL of 1 mg/mL proteinase K
6.After the 37В°C Incubation, add 20 pL of 5 mM Pefabloc SC, 4 pL 5 mg/mL
thyroglobulm, and 4 vol methanol
7 After 1 h at -2OвЂќC, the tube 1s centrifuged for 15 mm at 11 ,OOOg
8 Sonicate and boil the pellet mto 20 pL of sample buffer Analyze by SDS-PAGE-
fluorography
Usmg these condltlons, newly converted PK-reslstant 35S-PrP bands often
can be detected with an overnight fluorographic exposure of the gel Smce PK
treatment of scraple brain-derived PrP-res generally results m a 6-7 kDa
reduction in apparent molecular mass, we look for PK-resistant 35S-PrPbands
that are 6-7 kDa smaller than the corresponding untreated 35S-PrP-sen precursor
(52-H). The presence of 35S-PrPbands that are the same size as the 35S-PrP-sen
precursor would suggest that some nonspecific trapping or protection of
35S-PrP-sen precursor from PK may have occurred. We have also observed
PK-resistant 35S-PrP bands that are >6-7 kDa smaller than the 35S-PrP-sen
precursors, which has suggested that a partial conversion occurred (52,53).
The relationship between these incomplete cell-free conversion products and
brain-derived PrP-res molecules IS not clear. To control for the role of PrP-res
m the conversions, comparisons can be made to samples of 35S-PrP-senmcu-
bated without PrP-res or samples to which PrP-res IS added immediately before
the PK dtgestion (52,53) The efficiency of conversion of hamster 35S-PrP-sen
to PK-resistant species using the above protocol with hamster PrP-res has been
variable m our hands. Usually 5-30% of the 35S-PrP-senprecursor m the reac-
tlon becomes PK-resistant but, on occasion, as much as 100% of the label IS
converted. The reason for this varlabihty is not yet understood.
3 1.3. Specres and Strain Speaficities
in the Cell-Free Conversion Reaction
The cell-free conversion reaction can be used to investigate the species
specificity of PrP-res-PrP-sen mteractlons. For instance, mouse and hamster
PrP-res have been combined in conversion reactions with mouse, hamster, and
chimeric PrP-sen molecules (53). The species speclficitles in the conversion
PrP Metabolrsm and PrP-res Formation 295